Method of treatment employing therapeutic t cell product from mobilised donors

ABSTRACT

The present disclosure provides a method of treating a human patient in need thereof with immune reconstitution therapy by administering a therapeutically effective amount of therapeutic T cell population selected and/or expanded from a mobilised blood sample or a mobilised apheresis sample, wherein selection is on the basis of a steady state marker and/or an activation marker optionally followed by expansion, or expansion is in the presence of antigen, such as a viral antigen. It also extends to methods of generating said therapeutic T cell populations and the product obtainable therefrom.

The present disclosure relates to methods for preparing a T cellimmunotherapy product from a mobilised blood sample, for example forimmune reconstitution, the T cell population obtained from the saidmethod and pharmaceutical formulations comprising same. The disclosurealso provides the T cell population and said pharmaceutical formulationfor use in therapy, particularly immune reconstitution therapy, forexample in the treatment or prophylaxis of viral infections such as CMVand adenovirus infections.

BACKGROUND

Immune compromised patients are susceptible to opportunistic virusinfection. This is a huge problem in bone marrow transplant patientsbecause their immune cells are sometimes intentionally depleted as partof the bone marrow transplant procedure and other times renderednon-functional due to steroid treatment for Graft versus Host Disease(GvHD) which is a common complication of bone marrow transplantation.Latent viruses, such as CMV and adenoviruses, become re-activated andthe body is unable to fight the infection.

A practice of immune reconstitution has developed and this involves thetransplant (adoptive transfer) into the transplant patient of immunecells from a matched HLA donor, usually the same donor who provided thebone marrow or peripheral stem cell transplantation. These cells appearto engraft in the patient to provide long-term immunity to pathogens orat least interim assistance in fighting infection until the patient'sown immune system is fully reconstituted through the engraftment of thedonor's haematopoietic stem cells which will then develop into a diversearray of blood cells and immune cells.

Years of clinical research into the adoptive transfer of donor immunecells to achieve immune reconstitution in a patient following a bonemarrow transplant has illustrated the benefits of this approach as wellas the challenges of optimising the approach to ensure a consistentlyefficacious and safe result. In some cases, the number of donor immunecells which are necessary to effect immune reconstitution against aspecific pathogen cannot be obtained through simple mechanical selectionsystems. In such cases, the minimum dosing of the therapeutic immunecells, in particular antigen-specific T cells which demonstrate anadaptive memory immune response against the target pathogen, can beobtained by expanding the desired donor T cell population on an ex vivobasis using a cell culture system. Prior art indicates that the processof expansion of the cells from the donor sample generally takes about 21days and the focus has been to expand the specific cells in order toobtain the highest number possible number (yield) of the relevant cellpopulations as well as the highest possible purity of the relevant cellpopulations, for example to obtain a population which is as close to 100percent of the desired cells as possible.

The starting population of cells is obtained from a donor derived bloodsample or dedicated apheresis product. The current practice is that theapheresis product is harvested in a dedicated apheresis when the donorhas not undergone GCSF treatment and therefore is not a mobilised bloodsample, for reasons discussed below.

Mobilisation by recombinant human granulocyte-colony stimulating factor(G-CSF) is used to increase the number of donor stem cells incirculation prior to donation. This allows peripheral blood stem cellplant transplantation as opposed to bone marrow transplantation.Peripheral blood stem cell transplantation has a number of advantagesover bone marrow transplantation.

The current practice is that after a stem cell transplantation from thedonor to the patient and the donor is then required to return at afuture point in time, when the effects of the mobilisation havesubsided, to provide a further unmobilised blood sample or apheresiswhich can be used to generate a therapeutic T cell product to augmentthe patient's immune responses. The T cell product may be selected froma subset of cells from the sample and/or be expanded from a fraction ofa blood or apheresis sample.

Having to return for a second procedure is very inconvenient for donorsand can result in non-compliance which means that sometimes a bloodsample or leukapheresis is not available for generating an expanded Tcell product to augment the patient's immune responses.

Currently mobilised blood is not used to generate an expanded T cellproduct because early work established that mobilised blood does nothave the same properties as non-mobilised blood and in particular thatthere may be reduced activity in T cells in mobilised blood, for exampleMielcarek et al in Blood, Mar. 1, 1997 vol. 89 no. 5 1629-1634 describethe suppression of alloantigen-induced T-cell proliferation by CD14⁺cells derived from granulocyte colony-stimulating factor-mobilisedperipheral blood mononuclear cells.

In short after G-CSF stimulation in vivo, human and murine T cells showa reduced cytotoxic activity. A reduced proliferative response is alsoobserved upon in vitro stimulation.

Reyes et al in the British Journal of Cancer (1999) 80 (1/2), 229-235describes how granulocyte colony-stimulating factor (G-CSF) transientlysuppresses mitogen-stimulated T-cell proliferative response.

Murine and human studies have suggested that G-CSF mobilization inhibitstype 1 cytokine production by T cells, through inhibition of secretionas a single cell level as well as reducing the fraction ofcytokine-secreting cells in the periphery; arguing against the use ofthese cells in adoptive immunotherapy (Pan et al 1999, Arpinati et al200 and Tayebi et al 2001).

This reduced functionality of certain cells from mobilised-blood wasconfirmed by a number of authors see for example Joshi et al—Decreasedimmune functions of blood cells following mobilization with granulocytecolony-stimulating factor: association with donor characteristics Blood,15 Sep. 2001 Vol 98, No 6, 1963-1970, and Nawa et al G-CSF reduces IFN-Yand IL-4 production by T cells after allogeneic stimulation byindirectly modulating monocyte function, Bone Marrow Transplantation(2000) 25, 1035-1040.

G-CSF was also considered to have a role in immune tolerance, see forexample Anke Franzke's review in Cytokine & Growth Factor Reviews 17(2006) 235-244 entitled the role of G-CSF in adaptive immunity andRutella et al granulocyte colony-stimulating factor: a novel mediator ofT cell tolerance, The Journal of Immunology 2005 7085-7097. Whist immunetolerance to transplanted cells is desirable general immune tolerance isnot desirable when generating a therapeutic T cell product foraugmenting a patient's immune response. In fact tolerance may have somelinks with T cell anergy or hyporesponsiveness.

Other research has suggested that G-CSF may skew the T cell populationto the Th2 group, which may be less effective in controlling anintracellular viral infection.

Thus the practice in the field is to not employ mobilised-blood for thepreparation of expanded T cell products.

The present inventors believe that whilst in vitro T cells frommobilised blood appear less able to secret interferon-gamma (anactivation marker for antigen-stimulated T cells) as per FIG. 6 thecells nevertheless are suitable for use as T cell therapeutic product.This observation is somewhat counter-intuitive because interferon-gammais a pro-inflammatory cytokine involved in immune responses and askilled person would naturally consider that lower levels of secretionof this cytokine was indicative of generally lower activity of the Tcell from mobilised blood. However, it is possible to select and expandantigen specific T cells from mobilised blood and once taken out of themobilised cell environment these cells are not inferior in function to Tcells from non-mobilised blood.

Surprisingly, the present inventors have established that in facttherapeutic T cell products selected and/or expanded from G-CSFmobilised blood or mobilised apheresis are safe and effective whenadministered in vivo to a post-haematopoietic stem cell transplantpatient.

SUMMARY OF INVENTION

In one embodiment there is provided a method of treating patient in needthereof with immune reconstitution therapy by administering atherapeutically effective amount of a therapeutic T cell populationselected and/or expanded from a G-CSF mobilised blood sample ormobilised apheresis, in particular where the patient ispost-haematopoietic stem cell transplantation.

The present disclosure also provides a therapeutic T cell populationselected and/or expanded from a G-CSF mobilised blood sample or for usein treatment, in particular the treatment of a post-haematopoietic stemcell transplant patient.

In one embodiment the therapeutic T cell population is anantigen-specific T cell population.

In one embodiment the antigen-specific T cell population Is specific fora virus for example selected from the group comprising cytomegalovirus,adenovirus, varicella zoster virus, human papillomavirus, hepatitis Bvirus, hepatitis C virus, BK virus, Epstein-Barr virus, Kaposi'ssarcoma-associated herpes virus and human T-lymphotropic virus, such ascytomegalovirus or adenovirus.

In one embodiment the virus is cytomegalovirus.

In one embodiment the therapeutic T cell population is suitable fortreating virus infection, in particular a specific virus infectiondescribed herein or a combination of the same.

In one embodiment the T cells are allogeneic i.e. derived from a HLAmatched donor, in particular a fully matched donor.

In one embodiment the T cell population is selected on the basis of asteady state marker, for example the T cell receptors (TCR).

In one embodiment the T cell population is selected on the basis of amarker for example a marker that is independently selected from CD25,CD69, CD137, and CD154 and a combination thereof, for example CD69,CD137, and CD154 and a combination thereof, such as CD154.

After selection the cell population may be expanded to increase the doseof cells available for the patient.

Alternatively, a starting population of cells may be expanded in thepresence of antigen. This process involved a natural selection elementin that the process specifically cultivates cells specific to theantigen and non-target cell populations are reduced or eliminated.

In one embodiment the population of T cells does not comprisesignificant amounts of the cell surface marker CD25.

In one embodiment the therapeutic T cell product is selected from aG-CSF mobilised apheresis.

In one embodiment the therapeutic T cell product is expanded from aG-CSF mobilised blood sample.

Cells derived from mobilised sample may show reduced levels ofinterferon-gamma secretion in vitro. Nevertheless the inventors haveevidence to suggest that these cells are functioning and are suitablefor use in the therapeutic product despite in vitro property. Thisgenerates a practical difficulty in relation to the selection of therelevant populations because selections of the relevant T cellpopulations based on methods such as gamma-capture are sub-optimal.Therefore, if selection is to be employed a steady state T cell markerand/or an activation marker has to be employed. In one embodiment thisemploys a stimulation step followed by selection on a cell surfacemarker such as CD154, in another embodiment this employs a directselection method such as one based on the T cell receptor-streptamerselection.

Thus in fact mobilised-blood is a suitable starting material for thepreparation of T cell products and also provided is a method ofselecting and/or expanding a target T cell population which is specificto a virus from a starting T cells population from a mobilised bloodsample wherein selection employs direct selection targeting a steadystate maker on the surface of the T cells and expansion employsconditions suitable for expansion of target virus specific T cellpopulation.

Given the negative disclosures in relation to the use of mobilised-bloodsamples for the preparation of T cell products, it is very surprisingthat the material can in fact be employed successfully. Additionally themethods according to the present disclosure provide a huge advantage todonors, patients and healthcare workers because use of mobilised bloodsamples ensures the expanded T cell therapy will be available to morepatients without the inconvenience and disadvantages caused to donors bythe prior art methods.

There are also significant resource savings associated with the presentmethod because the collection, transport and storage of a second samplerequires a significant amount of additional human and financialresources.

Furthermore, being able provide an immunotherapy with T cells frommobilised blood may have the further advantage that the therapeuticproduct can be prepared immediately after the donation thereby avoidingthe “lag-time” associated with obtaining an unmobilised sample and thenprocessing the same to provide a therapeutic product.

Finally the donor has not to undergo an additional medical interventionand is therefore not put at the risks associated with an additionalleukapheresis procedure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Functional profile in unpaired G-CSF mobilised (n=6) andnon-mobilised (n=6) donors Quantitative assessment of IL-2, TNF, IFN-γ,IL-10, IL-4 and IL-5 in the supernatant of cultures after 16 hourCMVpp6S stimulation. Concentration of cytokine is expressed as a netvalue after subtraction of the negative control (unstimulated).

FIG. 2 Identification and isolation of IFN-g secreting antigen-specificT cells in unpaired G-CSF mobilised (n=6) and non-mobilised (n=6)donors. (2A) PBMC were stimulated for 16 hours with CMVpp65 and thefrequency of IFN-γ secreting cells analysed amongst CD3+ T cells. (2B)IFN-γ secreting cells were isolated using magnetic cell sorting andpurity and yield determined within the CD3+ population.

FIG. 3 Optimal time of expression of activation markers in response toCMVpp65 stimulation in G-CSF mobilised (n=5) and non-mobilised (n=5)PBMC. PBMC were stimulated over 24 hours and samples analysed for CD25,CD69 CD154 and CD137 expression at 1, 4, 6, 16 and 24 hours. IFN-γsecretion was analysed at 16 hours. Bars represent net expression in theCD3+ population for each activation marker at the optimal time ofexpression.

FIG. 4 Direct comparison between a G-CSF mobilised and non-mobiliseddonor of CD154 surface expression at 4 and 6 hours. (4A) PBMC werestimulated with either CMVpp65 Peptivator or SEB in the presence orabsence of CD40-specific antibody (1 μg/ml). Cells are gated on CD3+CD4+T cells (4B, C) Comparison of CD154 expression in non-mobilised (n=5)and G-CSF mobilised (n=5) donors. Data are presented as means withstandard deviation (SD)

FIG. 5 Isolation of CMV-specific T cells through CD154 expression in twounpaired donors. (4A) PBMC from non-mobilised and G-CSF mobilised donorswere stimulated with CMVpp65 Peptivator for 6 hours in the presence ofCD40-specific antibody. Cells were stained for CD154 amongst CD3+lymphocytes before stimulation after stimulation and after sorting ofCD154+ T cells on the MiniMACS. (B) Positive fractions from CD154+ sortsafter CMVpp65 stimulation in G-CSF mobilised (n=4) and non-mobilised(n=4) PBMC. Data are presented as means with SD.

FIG. 6 Re-stimulation of expanded CD154+ T cells. (A) Expanded CD154+ Tcells stained for CD3 and CD4 before re-stimulation after 21 days inculture. (B) Expanded CD154+ were co-cultured with autologous PBMC withor without CMVpp65 for 6 hours in the presence of CD40-specificantibody. After stimulation we analysed expression of CD154 versus CD69.(C) CD154+ expanded cells from G-CSF mobilised (n=3) and non-mobilised(n=3) PBMC analysed for CD154, CD69 (D) Expanded CD154+ from G-CSFmobilised PBMC were stimulated as described in the presence of BrefeldinA and CD28-specific antibody. Cells were fixed and permeabilised andanalysed for expression of CD154 versus IL-2, TNF and IFN-γ. (E)Analysis of IL-2, IFN-γ, and TNF expression after re-stimulation withautologous CMVpp65 PBMC in G-CSF mobilised (n=3) and non-mobilised (n=3)PBMC. Data are presented as means with SD.

FIG. 7 CD154+CMV-specific T cells isolated from G-CSF mobilised PBMCeffectively kill target cells. Specific lysis of autologous PHA blastsloaded with CMVpp65 peptides at E:T ratios from 50:1 to 0.5:1 determinedusing fluorescent dye Calcein-AM cytotoxicity assay.

FIG. 8 A sample of mobilised apheresis product was expanded for 10 daysusing a rapid expansion process—employing the G-rex40 culture device andIL-4 and IL-7. Cells were then re-stimulated with media alone(un-stimulated) or with CMV pp65 peptides. The amount of IFN gammaproduction was measured by flow cytometry. Cells are gated on livelymphocytes and CD3. This plots shows in Q1 that the desired populationof expanded cells from mobilised blood are capable of secretinginterferon-gamma. The skilled person will know that the profileexhibited in this plot is comparable to the profile obtained under thesame conditions for cells expanded from non-mobilised blood.

FIG. 9 A sample of mobilised apheresis product was expanded for 10 daysusing a rapid expansion process—employing the G-rex40 culture device andIL-4 and 11-7. Cells were then re-stimulated with media alone(un-stimulated) or with ADV Hexon V peptides. The amount of IFN gammaproduction was measured by flow cytometry. Cells are gated on livelymphocytes and CD3. This plots shows in Q1 that the desired populationof expanded cells from mobilised blood are capable of secretinginterferon-gamma. The skilled person will know that the profileexhibited in this plot is comparable to the profile obtained under thesame conditions for cells expanded from non-mobilised blood.

FIG. 10 A sample of mobilised apheresis product was taken from the stemcell harvest and sent to Cell Medica for processing. The cells wereexposed to the specific streptamer selection reagent and selected usingthe CliniMACS. This was then dosed at 3×10e4 T cells per Kg foradministration to the patient. The percentage of cells expressing theCMV specific T cell receptor (streptamer positive) was measured by flowcytometry. This shows that T cells can be successfully derived frommobilised apheresis samples in doses and purity equivalent tonon-mobilised products and can be administered to patients safely.

FIG. 11 Shows cells from FIG. 10 were gated on live lymphocytes and CD3.

FIGS. 12 & 13 Show that antigen specific T cells are functional evenwhen derived from an original sample which is mobilised

FIG. 14 Shows analysis of a sample therapeutic T cells selected bygamma-capture used to treat a patient with refractory CMV infection andthe starting material from which it was derived

DETAILED DESCRIPTION

Mobilised blood as employed herein refers to a blood sample from a donorwho has been mobilised by treatment with agent such as G-CSF. Theprocess of mobilisation increases the number of stems cells in theperipheral blood.

Apheresis as employed herein is the product of the process where theblood of a donor is passed through an instrument that separates out oneof more particular components from the blood and returns the remainderback into the donor's circulation.

Apheresis is employed to generate the leukapheresis employed in stemcell transplantation.

In preparation for the stem cell transplantation the leukapheresisproduct may undergo a selection for CD34+ stem cells. A bi-product isobtained from this process known as the CD34⁻ fraction.

Advantageously, the present process can employ this bi-product toselected or expand the therapeutic T cell population from. In oneembodiment the apheresis is a CD34− fraction.

Apheresis is also advantageous in that it potentially gives access to alarge number of cells in the starting material, for example in theregion of 1 to 10 billion cells, such as 2, 3, 4, 5, 6, 7, 8 or 9billion cells. This number of cells is sufficient to generate a suitabletherapeutic dose of T cell by selection only, i.e. without therequirement for subsequent expansion.

In contrast a blood sample may only contain in the region of 20 millioncells. Therefore if the starting material is a blood sample or a samplecontaining relatively low number of cells then an expansion step willgenerally be required generate a suitable therapeutic dose of cells forthe patient.

Mobilised apheresis as employed herein refers to a sample from a donorwho has been mobilised by treatment with agent such as G-CSF. Theprocess of mobilisation increases the number of stems cells in theperipheral blood.

There are various permutations of the present process and these aresummarised below:

-   -   1. Direct selection based on a steady state marker, such as the        TCR marker, to give a therapeutic dose (starting material an        apheresis),    -   2. Selection based on an activation marker, such as described        herein, to give a therapeutic dose (starting material an        apheresis),    -   3. Direct selection based on a steady state marker, followed by        expansion to give a therapeutic dose (starting material blood or        apheresis)    -   4. Selection based on an activation marker, followed by        expansion to give a therapeutic dose,    -   5. Expansion in the presence of antigen to generate a        therapeutic dose of an antigen specific T cell population        (starting material blood or apheresis).

Immune reconstitution as employed herein is intended to refer toproviding the host with a mechanism for generating an immune response oraugment the host's immune response to approximate that in a healthyindividual where otherwise the host's response would be minimal ornon-existent due to an impairment

In one embodiment the haematopoietic stem cell transplantation isallogeneic haematopoietic stem cell transplantation (allo-HSCT)including procedures involving stem cell donation from related orunrelated donors or from cord blood, such as peripheral stem celltransplantation unless the context indicates otherwise.

Effective in treatment as employed herein refers to a therapy that issafe for administration to patients and is at least broadly comparableto prior art T cell therapies derived from non-mobilised blood.

Therapy in the context of the present disclosure includes prophylactictherapy, which in the context of immune reconstitution is standardpractice.

“T cell” is a term commonly employed in the art and intended to includeall CD3+ cells including thymocytes, immature T lymphocytes, mature Tlymphocytes, resting T lymphocytes or activated T lymphocytes. A T cellcan be a T helper (Th) cell, for example a T helper 1 (Th1) or a Thelper 2 (Th2) cell, although other grouping of T cell populations arebeing discovered based on intensive research.

The T cell can be a CD4+ T cell, CD8+ T cell, CD4+CD8+ T cell, CD4-CD8−T cell or any other subset of T cells.

T cell product as employed herein refers to a population of T cellssuitable for use in therapy, for example immune reconstitution therapy.

Expanding a target T cell population as employed herein is intended torefer to increasing the number of the target cells in a population ofcells as a result of cell division, for example by culturing a startingpopulation of cells in a suitable medium.

T cell expansion may be evaluated by counting viable CD3+ cells (i.e.the target population of cells).

Viable cells can be tested by cell staining with Trypan blue (and lightmicroscopy) or 7-amino-actinomycin D, vital dye emitting at 670 nm (orViaProbe a commercial ready-to-use solution of 7AAD) and flow cytometry,employing a technique known to those skilled in the art. Where the stainpenetrates into the cells the cells are considered not viable. Cellswhich do not take up dye are considered viable. An exemplary method mayemploy about 5 μL of 7AAD and about 5 μL of Annexin-V (aphospholipid-binding protein which binds to external phospholipidphosphatidylserine exposed during apotosis) per approximate 100 μL ofcells suspension. This mixture may be incubated at ambient temperaturefor about 15 minutes the absence of light. The analysis may then beperformed employing flow cytometry. See for example M G Wing, A M PMontgomery, S. Songsivilai and J V Watson. An Improved Method for theDetection of Cell Surface Antigens in Samples of Low Viability usingFlow Cytometry. J Immunol Methods 126: 21-27 1990.

An alternative stain is TO-PRO-3 which is a carbocyanine monomer nucleicacid stain with far-red fluorescence similar to Alexa Fluor 647 or Cy 5dyes. It is useful as a nuclear counterstain and dead cell indicator,and is among the highest-sensitivity probes for nucleic acid detection.

In one embodiment the T cell population is selected from mobilised bloodby direct selection based on a steady state marker, such as the T cellreceptor (TCR). This process employs HLA:peptide complexes particularlyin the form of multimers, such as tetra, penta and/or hexamers whichligate to T cell receptor. These peptides are labelled, for example witha fluorescent label or a magnetic bead which allows then to them to beidentified and selected. In one embodiment a magnetic label is employed.

Thus direct selection generally involves the clinical grade enrichmentof lymphocytes from a fraction of mobilised apheresis product. This mayuse a dedicated device such as a Sepax device from Biosafe. Theresulting lymphocytes are then incubated with a selection reagent whichis a mulimerised MHC/peptide complex attached to a magnetic bead. Wherethe MHC/peptide complex is matched to the patient and donor and isspecific for an antigen specific T cell receptor. Following incubationthe cells are washed and the bound cells are selected with a device suchas the Miltenyi CliniMACS or any other technology that enables cellselection using magnetic beads where positive cells are retained on amagnetic column or in a bag and the negative cells are washed off. Themagnet is then removed and the antigen specific cells are eluted

In one embodiment the multimers are Streptamers. The ligation of the TCRby Streptamers is reversible and after selection of the desiredpopulation of cells then treatment with a specific reagent results isremoval of the complexes from the cells.

HLA complexes employed need to be matched with the HLA type so that theycan ligate a virus specific population of T cells, such that thepeptides or multimers are of a specific HLA-type, for example A1, A2,B7, A24, B35, such as A0201 and B0702.

Selection can also or alternatively be based on activation markers.These are makers which are upregulated as a consequence of antigenstimulation. A plethora of these exist and are known to those skilled inthe art and include makers such as CD25, CD69, CD137, CD154 andcombinations thereof.

These markers can be selected by ligation with monomeric, dimeric,multimeric antibody or binding fragments thereof. These antibodies orfragments are labelled, for example with a fluorescent label or amagnetic bead which allows then to them to be identified and selected.In one embodiment a magnetic label is employed.

In one embodiment the antibody or fragment employed is a fab-streptamer,for example available from IBA GmBH Germany.

The binding of these fab-streptamers is reversible in that afterselection of the desired cell population treatment with an appropriatereagent releases them from the cells.

Ligation as employed herein refers to binding.

In one embodiment the T cell population is selected from mobilised bloodand then expanded.

In one embodiment selection is not required before expansion because theexpansion selectively enriches for the target population of cells whichis facet of the expansion process.

A T cell population specific to a virus as employed herein is intendedto refer to the fact that the relevant population of cells primarilyrecognises and at least one viral antigen to which is specific, and forexample generates an immunological response after recognition of thetarget virus. Specificity in this context does not necessarily mean thatonly the target virus is recognised, although in some instances only thetarget virus will be recognised, but at least the target virus isrecognised with greater affinity, avidity or magnitude of response incomparison to non-target viruses.

Viral antigen as employed herein is intended to refer to those antigensspecified by the viral genome (often coat proteins) that can be detectedby a specific immunological response. In one embodiment the viralantigen is a surface antigen.

In one embodiment the virus is a DNA virus, for example a doublestranded DNA virus.

In one embodiment the virus is an RNA virus.

Typically the PBMCs are obtained from the blood or apheresis product byFicoll density gradient separation known to those skilled in the art.

The step of obtaining a sample from the patient can be a routinetechnique of taking a blood sample.

This process presents little risk to patients and does not need to beperformed by a doctor but can be performed by appropriately trainedsupport staff. In one embodiment the sample derived from the patient isapproximately 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 50 ml, 40 ml, 30ml, 20 ml, 10 ml, 5 ml or less of blood.

In one embodiment the starting material is a fraction of the mobilisedapheresis product that is taken once it has been ensured that the CD34+cell dose for the patient has been achieved. For example 4×10e6 CD34+cells per kg patient weight.

In one embodiment the cells which are bi-product of the stemtransplantations are employed. Stem cells for transplantation are oftenselected on the basis of CD34. Those populations which are negative forCD34 are often discarded after selection. However, this deselectedpopulation is suitable for generating a therapeutic T cell product, forexample employing a method described herein, such as T cell expansion.

As is known to the skilled person expansion of T cells is generallyperformed in a suitable T cell expansion media. T cell expansion mediagenerally comprises serum, media and any cytokines employed in theexpansion step.

In one embodiment the media is Advanced RPMI media or RPMI media 1640,available from Life Technologies.

In one embodiment the cell expansion medium comprises 10% human ABserum, 200 mM L-glutamine, and RPMI-1640.

In one embodiment the medium comprises 45% advanced RPMI, 45% EHAA, 10%FCs and 200 mM L-glutamine.

In one embodiment the cell expansion medium comprises 10% human ABserum, 200 mM L-glutamine, 45% Earle's Ham's amino acids (EHAA orClick's medium) and 45% advanced RPMI or RPMI-1640.

In one embodiment the cytokines employed are discussed below.

In one embodiment the T cell expansion medium employed is not changed orsupplemented during the expansion process, especially where a rapidexpansion process is employed. Rapid expansion as employed herein refersto a process in a therapeutic product is obtained within less than 18days, such as 7-10 days.

Thus in one embodiment there is provided according to the presentdisclosure an in vitro expansion process for rapid expansion of antigenspecific T cells (such as allogeneic antigen specific T cells)comprising the steps culturing in a gas permeable vessel a population ofPBMCs (such as allogeneic PBMCs) in the presence of a peptide or peptidemix relevant to a target antigen(s), in the presence of an exogenouscytokine characterised in that the cytokine is other than exogenousIL-2.

In one embodiment there is provided according to the present disclosurein vitro expansion process for rapid expansion of antigen specific Tcells, such as allogeneic antigen specific T cells comprising the stepsculturing in a gas permeable vessel a population of PBMCs (such asallogeneic PBMCs) in the presence of antigen, for example a peptide orpeptide mix relevant to a target antigen(s), in the presence of anexogenous cytokine characterised in that the expansion to provide thedesired population of T cells is 14 days or less, for example 9, 10, 11or 12 days, such as 10 days.

Cytokines that may be employed in the process of the current disclosureinclude IL-1, IL-2, IL-4, IL-6 IL-7, IL-12 and IL-15.

A large amount of, as yet non-definitive, literature underlines howIL-2, IL-7 and IL-15 play non-redundant roles in shaping therepresentation of memory cells. IL-2 controls T-cell clonal expansionand contraction, and promotes lymphocyte differentiation. IL-2 and IL-15can also support memory cell division and have been used in combinationwith antigen-driven stimulation, for the expansion of CTL IL-7 regulatesperipheral T-cell homeostasis, and contributes to the generation andlong-term survival of both CD41 and CD81 memory T lymphocytes in vivo.

In one embodiment the cytokines employed in the expansion processaccording to the present disclosure are independently selected fromIL-4, IL-7 and IL-15, especially IL-4 and IL-7.

In one embodiment the cytokines employed are IL-4 and/or IL-7. Whilstnot wishing to be bound by theory the inventors believe that thesecytokines have a role to play in shaping the frequency, repertoire andexpansion of viral antigen-specific T cells.

In one embodiment the method according to the present disclosureprovides a T cell population which has a repertoire of antigen-specificT cells.

The repertoire of T cells may be determined by ELISPOT analysis afterstimulation with peptide libraries aliquotted into pools such that eachpeptide is uniquely represented in two pools (Kern, F., N. Faulhaber, C.Frommel, E. Khatamzas, S. Prosch, C. Schonemann, I. Kretzschmar, R.Volkmer-Engert, H. D. Volk, and P. Reinke. 2000. Analysis of CD8 T cellreactivity to cytomegalovirus using protein—spanning pools ofoverlapping pentadecapeptides. Eur J Immunol. 30:1676-1682 andStraathof, K. C., A. M. Leen, E. L Buza, G. Taylor, M. H. Huls, H. E.Heslop, C. M. Rooney, and C. M. Bollard. 2005. Characterization oflatent membrane protein 2 specificity in CTL lines from patients withEBV-positive nasopharyngeal carcinoma and lymphoma. J. Immunol.175:4137-4147) or by intracellular cytokine staining by plating 200,000of the final T cell product in a round bottomed 96 well plate and usingpeptides as above to re-stimulate the cells at a concentration of 1ug/ml. This is performed overnight in the presence of 5 ug/ml ofBrefeldin A which prevents secretion of cytokine and therefore is usedto ensure build-up of IFNg inside the cells for enumeration using flowcytometry.

IL-4 is generally employed at a final concentration of 250 ng/ml ofculture or less, such as 200 ng/ml or less.

IL-7 is generally employed at a final concentration of 50 ng/ml ofculture or less, such as 20 ng/ml or less, in particular 10 ng/ml.

If 11-15 is employed a suitable final concentration is 50 ng/ml ofculture or less, such as 20 ng/ml or less, in particular 10 ng/ml.

In one embodiment in about 20 mls per GRex-10 (for example 20×10⁶ PBMCs)a further 10 mls medium containing IL-4 (1666 units per mL) and IL-7 (10ng per ml) is added.

IL-12 has a role in Th1 focussing and exogenous IL-12 may be omitted ifa balanced Th1/Th2 is desired. In one embodiment the process of thepresent disclosure does not employ exogenous IL-12. However, in thecontext of the present T cell product a Th1 response in the CD4+population is thought to be desirable.

In one embodiment when IL-4 is employed the in the expansion process ofthe present disclosure. At day 10 or day 11 the number of expanded cellsmay be 10, 20, 30, 40 50, 60, 70, 80, 90, 100 or 200% higher than cellsexpanded employing a similar protocol replacing IL-4 with IL-2.

When exogenous IL-2 is employed in the rapid expansion systemhyper-proliferation of T cells is generated. When this hyper-rapidexpansion occurs then the balance of desirable T cells and the residualcells is suboptimal in that the expansion happens so rapidly that manythat the residual cells have not died and thus remain present in thetotal cell population. Thus the present inventors have reconciled theinherently incompatible factors of rapid expansion with the selectivityof culturing the cells for a period of time which allows death of thenon-target cells and have found that the omission of IL-2 improves theratio of desired cells to residual cells. What is more in the period 7to 14 days such as 10 days the ratio of desired cells to residual cellsis a cross-over point where the cultured product becomes suitable foruse in therapy. This cross-over point is defined as when a sufficientminimum dose of therapeutic T cells is achieved within a doseformulation which falls within the safety threshold of no more than5×10⁵ CD3+ T cells per kg of patient body weight.

In one embodiment the T cell population as allogeneic, that is to saythe T cell population is derived from a donor who is not the patient.

Generally the donor will be fully HLA matched.

The human leukocyte antigen (HLA) system is the name of the majorhistocompatibility complex (MHC) in humans. The super locus contains alarge number of genes related to immune system function in humans. Thisgroup of genes resides on chromosome 6, and encode cell-surfaceantigen-presenting proteins and many other genes. The HLA genes are thehuman versions of the MHC genes that are found in most vertebrates (andthus are the most studied of the MHC genes). The proteins encoded bycertain genes are also known as antigens, as a result of their historicdiscovery as factors in organ transplants. The major HLA antigens areessential elements for immune function. Different classes have differentfunctions:

HLAs corresponding to MHC class I (A, B, and C) present peptides frominside the cell (including viral peptides if present). These peptidesare produced from digested proteins that are broken down in theproteasomes. In general, the peptides are small polymers, about 9 aminoacids in length. Foreign antigens attract killer T-cells (also calledCD8 positive- or cytotoxic T-cells) that destroy cells.

HLAs corresponding to MHC class II (DP,DM, DOA,DOB,DQ, and DR) presentantigens from outside of the cell to T-lymphocytes. These particularantigens stimulate the multiplication of T-helper cells, which in turnstimulate antibody-producing B-cells to produce antibodies to thatspecific antigen. Self-antigens are suppressed by suppressor T-cells.

In one embodiment the selection of cells is based on theinterferon-gamma secretion or a cell surface activation marker, afterstimulation of the cells with antigen, in particular peptides of arelevant antigen.

In one embodiment the mobilised blood sample obtained from the donor maybe cryopreserved before processing.

In one embodiment after expansion optionally one or more components suchas stabilising agents and/or cryopreservants are added to theformulation, for example human serum albumin, glycerol, DMSO or similar.

The present invention also extends to compositions comprising theallogeneic antigen-specific T cell populations according to theinvention. These compositions, may comprise a diluent, carrier,stabilizer, surfactant, pH adjustment or any other pharmaceuticallyacceptable excipient added to the cell population after the main processsteps. An excipient will generally have a function of stabilizing theformulation, prolonging half-life, rendering the composition morecompatible with the in vivo system of the patient or the like.

In one embodiment a protein stabilizing agent is added to the cellculture after manufacturing, for example albumin, in particular humanserum album, which may act as a stabilizing agent. The amounts albuminemployed in the formulation may be 10 to 50% w/w, such as about 12.5%w/w.

In one embodiment the formulation also contains a cryopreservative, forexample glycerol or DMSO. The quantity of DMSO is generally 12% or lesssuch as about 10% w/w.

In one embodiment the process of the present invention comprises thefurther step of preparing a pharmaceutical formulation by adding apharmaceutically acceptable excipient, in particular an excipient asdescribed herein, for example diluent, stabilizer and/or preservative.

Excipient as employed herein is a generic term to cover all ingredientsadded to the T cell population that do not have a biological orphysiological function.

In one embodiment the pharmaceutical composition is adapted foradministration by infusion.

In one embodiment the target virus to which an antigen specific T cellpopulation is generated is CMV and, for example the antigen employed tothe target the virus is pp65. The sequence for human cytomegalovirus(strain AD169) is in the UniProt database under number P06725. Therecombinant protein can be purchased from Miltenyi Biotech. The lattercompany also provide PepTivator® CMV pp65 which is a peptide pool thatconsists mainly of 15-mer peptides with 11-amino acid (aa) overlap,covering the complete sequence of the pp65 protein of humancytomegalovirus.

In aspect the disclosure extends to a T cell product obtained orobtainable from the present method.

In one aspect the disclosure extends a virus specific expanded T cellproduct

In one embodiment the disclosure extends treatment or prophylaxis of apatient with a T cell product according to the present disclosure or acomposition comprising the same 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeksor more after receiving a bone marrow transplant or peripheral stem celltransplant.

All citation and documents referred herein are specifically incorporatedby reference. All references to literature and patent documents areincorporated by reference.

Comprising in the context of the present invention means including.

Described above are embodiments comprising certain integers. Embodimentsof the invention described above can be combined as technicallyappropriate. The present disclosure also extends to correspondingembodiments consisting of said integers as herein described.

EXAMPLES Example 1 Blood Donors and Cell Preparation

We obtained blood samples from both G-CSF mobilised and non-mobilisedhealthy donors after a 3-5 hour leukapheresis. Informed consent wasobtained in accordance with the Declaration of Helsinki and studies wereapproved by the Royal Free NHS Trust Research and Development reviewboard. Peripheral blood mononuclear cells (PBMC) were generated usingFicol (Axis Shield Diagnostics) density gradient separation and culturedin RPMI 1640 Medium (Gibco) supplemented with 1% antibiotic (Gibco) and10% heat inactivated human AB serum (Biosera) at a concentration of1×10⁷/ml. Excess PBMC were cryopreserved at 1:1 with human serum albumin(HSA) 4.5% (Bio Products Laboratory) containing 20% DMSO (WakChemie) asa future source of feeder cells. PBMC were stimulated for up to 24 hoursin 6 well culture plates (Nunc) with CMVpp65 Peptivator (MiltenyiBiotec) at 37° C./5% CO₂. For CD154 experiments cultures were stimulatedin the presence of 1 μg/ml anti-CD40 antibody (BioLegend)

Flow Cytometry Analysis

Flow cytometry experiments consisted of four to six colour panels wherea minimum of 50,000 CD3+ were acquired after gating of viablelymphocytes using FSC and SSC signals on a FACScan flow cytometer (CytekUK) and data analysed using FlowJo version 7.6 (TreeStar). For isotypecontrol staining of cytokines and activation markers we usedPE-conjugated mouse IgG1 K antibodies (BD Bioscience). Cells werestained for 15 minutes in the dark, washed in 2 ml of HBSS for 5 minutesand resuspended in 200 μl of FACS Flow (BD Bioscience) beforeacquisition. Cytokine analysis of supernatants from CMVpp65 stimulatedand untouched PBMC were performed on a FACS Aria flow cytometer (BDBiosciences) and a minimum of 20,000 events collected.

Cytokine Analysis by Cytometric Bead Array (CBA)

Supernatants were collected from CMVpp65 stimulated and non-stimulatedcontrol cultures at 16-24 hours from both mobilised and non-mobiliseddonors and stored at −80′C. Analysis by Cytometric Bead Array Kit (BDBiosciences) was used to quantify the level of IL-2, IL-4, IL-5, IL-10,IFN-γ and TNF. Analysis of Acquired Data was Performed Using FCAP ArraySoftware Version 1.0.1 (Soft Flow Hungary Ltd.).

Time course Assay

PBMC isolated from mobilised and non-mobilised donors were stimulated in96 well plates at a concentration of 1×10⁷/ml for 24 hours with eitherCMVpp65 Peptivator or 1 μg/ml SEB (Sigma) or left untouched. Sampleswere taken at 1, 4, 6, 16 and 24 hours and stained with APC-conjugatedanti-CD3, FITC-conjugated anti-CD4, PerCP-conjugated anti-CD8 and eitherPE-conjugated anti-CD154, anti-CD25, anti-CD69 or anti-CD137 (all BDBioscience).

Isolation of Antigen Specific T Cells

For the isolation of antigen-specific T cells following CMVpp65stimulation cells were either stained with PE-conjugated anti-CD25 after16 hours or PE-conjugated anti-CD154 after 6 hours (both BD Bioscience).Labelling was performed for 20 minutes using 10 μl of antibody per 10⁷cells in 100 μl of CliniMACS buffer. After 20 minute incubation withPE-conjugated microbeads (20 μl/10⁷ cells) in 80 μl of CliniMACS bufferthe cell suspension was enriched using MS columns on a MiniMACS (allMiltenyi Biotec). All incubation steps were performed at 4-8° C. in thedark. Antigen-specific T cells were also isolated using the IFN-γsecretion assay according to the manufacturer's recommendation (MiltenyiBiotec) and isolation was identical to that of CD154 and CD25separation.

This was also performed on steady state specificity marders such as thespecific T cell receptor whereby Stage/IBA product—streptamers were usedas the selection reagent and on a clinical scale using the CliniMACS forselection.

Expansion of Antigen-Specific T Cell Lines

After a 6 hour incubation, we cultured up to 0.25×10⁶ isolated CD154+cells in the presence of 50:1 γ-irradiated (30 Gy) autologous PBMC toact as feeder cells in 24 well plates with RPMI 1640 medium containing10% human AB serum, 1% antibiotic and supplemented with 10 ng/ml of IL-7and IL-15 (Cell Genix). Culture medium was replenished every 2-3 daysand cells split when necessary. Cells were expanded up to a maximum of23 days before harvest.

Where cells were not selected prior to expansion, cells were seeded at2×10e6 PBMC per ml in 20 ml in the G-rex10 expansion system from WilsonWolf. The cells were seeded with the specific peptide, IL-4 and IL-7 inRPMI 10% human serum and were cultured un-touched for 10 days.

Re-Stimulation of Expanded Antigen-Specific T Cell Lines

We restimulated expanded cells for a period of 5-6 hours with eitherCMVpp65 Peptiavtor, CMV IE-1 (JPT) loaded autologous PBMC or untouchedautologous PBMC as a control, all labelled with 1 μM CFSE (Sigma) at aratio of 2.5:1 at a concentration of 1×10⁷/ml in 48 well plates. Foranalysis of intracellular cytokines and CD154 we incubated cells in thepresence of anti-CD28 antibody (BD Bioscience) and added 1 μg/ml ofBrefeldin A (Sigma) after 2 hours. Cells were fixed and permeabilisedusing Intrastain (DakoCytomation) according to the manufacturer'sinstructions and stained with APC-conjugated anti-CD154,PerCP-conjugated anti-CD4 either PE-conjugated anti-IL-2, anti-TNF oranti IFN-γ (all BD Biosciences). For surface staining cells wereincubated in the presence of anti-CD40 antibody and then stained for 10minutes with FITC-conjugated anti-CD4, PE-conjugated anti-CD154,PerCP-conjugated anti-CD8, APC-conjugated anti-CD3 and APCCy7-conjugated CD69 (all BD Biosciences).

Cytotoxicity Assay

Autologous PBMC were stimulated with 3 μg/ml of PHA (Sigma) for 24 hoursand then 20 U/ml of IL-2 (Miltenyi Biotec) at a concentration of1×10⁶/ml in RPMI 1640 with 10% AB serum. PHA blasts were then loadedwith CMVpp65 Peptivator to use as target cells. Loaded target cells werelabelled with Calcein-AM (Molecular Probes) at a concentration of 10 μMand incubated for 1 hour at 37′C. After four washes in complete mediumcells were adjusted to 7×10⁴/ml and added to effector cells at E:Tratios ranging from 20:1 to 0.5:1, in triplicate, in U bottom 96 wellplates (Corning). Triplicate wells were also set up to measurespontaneous release (target cells only), maximal release (target cellsplus 2% Triton X-100) and medium alone. After incubation at 37′C/5% CO₂for four hours, 100 μl of supernatant was harvested and transferred intonew plates. Samples were measured using a BMG FLUOstar Galaxy microplatefluorescence spectrophotometer (MTX Lab Systems Inc.) (excitationfilter: 485±9 nm: band-pass filter: 530±9 nm). Data were expressed asarbitrary fluorescent units (AFU) and percent lysis was calculated usingthe formula [(test release-spontaneous release/maximalrelease-spontaneous release)×100].

Statistical Analysis

Analyses were conducted using GraphPad Prism 4.0. The nonparametricMann-Whitney test was used to determine the statistical significancebetween G-CSF mobilised and non-mobilised PBMC and a Paired t test foranalysing the effect of CD40 blocking on CD154 expression. Statisticalsignificance was achieved when P was less than 0.05.

Results

Cytokine Profile of CMVpp65 stimulated G-CSF mobilised PBMC Initialexperiments aimed to investigate the cytokine profile of CMVpp65stimulated PBMC from G-CSF mobilised PBMC to determine whether there wasequivalence with non mobilised PBMC. PBMC from CMV+ healthy individualswere stimulated with CMVpp65 overlapping peptides in 16 hour cultures.

After 16 hours aliquots of supernatant were taken from stimulated anduntouched cultures and frozen at −80′C. Supernatants were assayed forthe cytokines released during the culture period using a flow cytometricbased assay, the cytokine bead array (CBA). IL-2, TNF, IFN-γ, IL-10,IL-4 and IL-5 secretion were analysed (FIG. 1). No significantdifference was observed between G-CSF mobilised and non-mobilised PBMCin terms of the TH: cytokines IL-2, TNF and IFN-γ, but a significantdecrease in IL-10 secretion from G-CSF mobilised PBMC (P=0.01) wasdetected and this trend was also evident in the low levels of IL-4 andIL-5 secretion.

Next we evaluated whether CMV-specific T cells could be isolated fromG-CSF mobilised PBMC based on IFN-γ secretion, as we have used thissystem previously for the manufacture of CMV-specific T cells fromnon-mobilised PBMC and demonstrated their clinical efficacy. Cellssecreting IFN-γ in response to CMVpp65 stimulation were captured usingIFN-γ specific antibodies and selected using magnetic beads.

IFN-γ was measured before and after magnetic enrichment to assess purityand yield between mobilised and non-mobilised PBMC. Although notsignificant we showed that IFN-γ secretion was decreased after CMVpp65stimulation (FIG. 2A) and that purity and yield (FIG. 2B) were alsonegatively affected in G-CSF mobilised PBMC. The ratio of CD4+ toCD8+IFN-γ secreting cells appeared to be unchanged in G-CSF mobilisedPBMC. In summary PBMC from G-CSF mobilised PBMC are capable of secretingIFN-γ and other effector cytokines at a level similar to non mobilisedPBMC, but isolation and detection after CMVpp65 stimulation on a percell basis appears to be impaired. These results are in line withpreviously published data suggesting that G-CSF mobilisation impairs thepotential for IFN-γ production at a single cell level.²⁵

Analysis of Activation Marker Expression after CMVpp65 Stimulation

We next investigated the kinetics of activation induced CD25, CD69,CD154 and CD137 expression on CMVpp65 specific T cells in G-CSFmobilised PBMC to determine the optimal duration of stimulation incomparison to non-mobilised PBMC. We stimulated PBMC over a 24 hourperiod with CMVpp65 peptides and removed PBMC populations from culturesat 1, 4, 6, 16 and 24 hours and then analysed for surface expression ofactivation markers by flow cytometry (FIG. 3). Antigen triggeredexpression of CD25 was optimal at 16 hours and was of the same intensityin G-CSF mobilised and non-mobilised PBMC. CD69 and CD154 were optimalat 6 hrs and expression of both was elevated in G-CSF mobilised PBMC.CD137 expression reached peak intensity at 24 hours and was alsoincreased in G-CSF mobilised PBMC. In line with previous results weobserved a reduction in the level of IFN-γ secretion from G-CSFmobilised PBMC at 16 hours after CMVpp65 stimulation.

Assessment of Antigen Specific Expression of CD154 in G-CSF MobilisedPBMC

Previously published data have demonstrated that CD154 is a suitablemarker for the detection and isolation of CMV-specific T cells. Wetherefore investigated whether CD154 expression in G-CSF mobilised PBMCwas consistent with non-mobilised PBMC, using a CD40-specific antibodyto preserve CD154 at the cell surface by preventing ligation with CD40.PBMC were stimulated with either SEB or CMVpp65 peptides for 4-6 hoursin the presence or absence of CD40-specific antibody, and then analysedfor CD154 expression amongst the CD4+ T cell population (FIG. 4A).

Low background CD154 expression in resting CD4+ T cells was comparablebetween G-CSF mobilised PBMC (0.30%) and non-mobilised PBMC (0.22%).CD154 expression in the presence of CD40-specific antibody at theoptimal time point of 6 hours, showed no statistical significantdifference between G-CSF mobilised PBMC (1.86%) and non-mobilised PBMC(1.22%) but was in fact elevated in the G-CSF mobilised donor setting(FIG. 4B-C), without any unspecific activation induced CD154 expression.

Isolation of Antigen-Specific T Cells from G-CSF Mobilised andNon-Mobilised PBMC Through CD154 Expression

We next performed a single enrichment step of CMVpp65 stimulated PBMC inthe presence of CD40-specific antibody from G-CSF mobilised andnon-mobilised PBMC (FIG. 5A-B) using magnetic cell separation in 4 CMV+healthy unpaired donors. We observed no significant difference in thepurity of CD154+CMV-specific T cells (FIG. 5C) between G-CSF mobilised(48.94%) and non-mobilised (58.08%) PBMC. CD154 positive fractions weresubsequently expanded in short term culture to determine in vitroproliferation and CMV specificity of isolated cells.

Re-Stimulation of In-Vitro Expanded Antigen-Specific from G-CSFMobilised PBMC

CD154+CMV-specific T cells were cultured over 21 days in complete mediumcontaining IL-7 and IL-15 in the presence of autologous irradiatedfeeder cells. CD154+ responder populations showed a mean amplificationfactor of 74.6-fold (range 48-84) in G-CSF mobilised PBMC (n=3) comparedto 103.6 (range 18-168) in non-mobilised PBMC (n=3). Expanded cells werepredominantly CD3+CD4+ in all cultures (FIG. 6A) All cultures showedhigh specificity for CMVpp65 determined by up regulation of CD154+ andCD69+ expression upon re-challenge with autologous CMVpp65 loaded PBMC.In control re-challenge experiments with autologous PBMC alone, low toundetectable levels of CD154 expression was observed (FIG. 6B). Weobserved an increase in the up-regulation of CD154+CD69+ expression uponre-challenge in cells expanded from G-CSF mobilised PBMC (mean, 93.13%)compared to non-mobilised PBMC (mean, 63.0%) after flow cytometricanalysis (FIG. 6C). In some experiments expanded cells werere-challenged with CMV IE-1 peptides and no CD154 activation wasobserved confirming specificity (data not shown).

To analyse the functionality of expanded cells we also tested forproduction of IL-2, TNF and IFN-γ by intracellular cytokine staining(ICS) (FIG. 6D). Expanded cells were capable of synthesising andsecreting all three cytokines, but predominantly IFN-γ. In experimentswhere expanded cells were unstimulated or incubated with CMV IE-1peptides, minimal cytokine secretion was observed. No significantdifferences were detected in IL-2, TNF or IFN-γ secretion between G-CSFmobilised PBMC and non-mobilised PBMC (FIG. 6E). We have demonstratedthat the CD154 assay allows for specific isolation of both expandableand functional CMV-specific T cells from G-CSF mobilised PBMC that isequivalent to published data in non-mobilised PBMC.

Cytotoxic Activity of Expanded Cells

Finally we investigated whether expanded CD154+CMV-specific T cellsisolated from G-CSF mobilised PBMC are able to lyse target cells.Autologous PHA blasts loaded with CMVpp65 peptides and labelled withCalcein-AM dye were used as targets. Targets were effectively killed byexpanded cells (FIG. 7) at all E:T ratios.

Example 2 Analysis of Cells Obtained from the Negative Fraction of CD34Selection

The starting material was the negative fraction from a CD34 selectionfrom mobilised HPC-A (also referred to herein as an apheresis sample).

Cells underwent density gradient centrifugation prior to being culturedfor 10 days with ADV peptide, IL-4 and IL-7. On day 10 cells wereharvested, washed, counted, dosed and cryopreserved. Potency testing forgamma production and phenotyping for purity and viability was alsoperformed.

Doses of 1×10⁴ and 1×10⁵ T cell per Kg were frozen (12 Kg).

7.56% of T cells produced IFNγ following re-stimulation with ADV peptide(release criteria states 1%) and all other release criteria (T cellpurity, viability, microbiology, mycoplasma, endotoxin) were met. Thescatter plot for this analysis is shown in FIG. 11.

PBMC derived from mobilised and non-mobilised material can be acceptedas starting material for the process.

No paired samples have been analysed however 9 production runs have beenperformed on each starting material. Below is a table showing the % IFNgproduction upon re-stimulation for both mobilised and non-mobilisedproduct

Mobilised product Non mobilised product 1.32 2.22 2.45 1.85 5.26 0.241.95 13.86 6.53 21.19 1.31 1.95 7.56 2.71 3.62 4.89 1.54 17.38 Mean 3.5Mean 7.3 SD 2.4 SD 7.9

Example 3

The data in FIGS. 12 and 13 show that antigen specific T cells arefunctional even when derived from an original sample which is mobilised.PBMC derived from mobilised apheresis were stained with CFSE—a dye thatis taken up by cells and when a cell divides the brightness of the cellsis reduced and this can be detected by flow cytometry. The cells werecultured for 5 days at 37 deg C. with either no stimulation (nil) orwith the antigen specific peptide, prior to being stained forstreptamer, CD3, CD8 and run on a flow cytometer. This shows that thecells can proliferate despite being mobilised as long as there is thesufficient stimulus and they will not proliferate unless the stimulus isthere—showing function.

Example 4 Treatment of a Patient

Cells were selected by gamma catch from a frozen mobilised apheresissample and were used for the treatment of a 72 Kg patient withrefractory CMV (at least 2 months) with CMV retinitis involvement. Adose of about 22,000 CMV specific T cells was administered by infusion.Following treatment CMV and retinitis resolved and the patient wasdischarged from hospital. Thus despite the mobilisation the cellsadministered were functional. The FIG. 14 shows that some gamma wasproduced in the pre selection population, it was reduced in the negativefraction and the positive fraction was the product that was actuallyadministered to the patient

1. A method of treating a human patient in need thereof with immunereconstitution therapy by administering a therapeutically effectiveamount of therapeutic T cell population selected and/or expanded from amobilised blood sample or a mobilised apheresis sample, whereinselection is on the basis of a steady state marker and/or an activationmarker optionally followed by expansion, or expansion is in the presenceof antigen, such as a viral antigen.
 2. A method according to claim 1,wherein the patient is post-haematopoietic stem cell transplantation. 3.A method according to claim 1, wherein the T cell population is anantigen-specific T cell population.
 4. A method according to claim 3,wherein the antigen-specific T cell population is specific a virus forexample selected from the group comprising cytomegalovirus, adenovirus,varicella zoster virus, BK virus, human papillomavirus, hepatitis Bvirus, hepatitis C virus, Epstein-Barr virus, Kaposi'ssarcoma-associated herpes virus and human T-lymphotropic virus, such ascytomegalovirus or adenovirus. 5-28. (canceled)
 29. A method accordingto claim 4, wherein the antigen-specific T cell population is specificto cytomegalovirus.
 30. A method according to claim 29, wherein theantigen-specific T cell population is specific to pp65.
 31. A methodaccording to claim 1, wherein the population is directly selected on thebasis of a steady state marker namely the T cell receptor, for exampleby reversible ligation of the T cell receptor by specific HLA:peptidecomplexes, in particular Tetra, Penta and/or Hexa streptamers.
 32. Amethod according to claim 1, wherein the activation marker is a cellsurface marker that is upregulated as a consequence of antigenstimulation, for example selected from the group comprising CD25, CD69,CD137 and CD154.
 33. A method according to claim 1, wherein the T cellpopulation is expanded from a mobilised blood sample or mobilisedapheresis sample, in particular expanded in an antigen specific manner.34. A method according to claim 1, wherein the population issubstantially negative for cells with the CD25 marker.
 35. A methodaccording to claim 1, wherein the T cell population is allogeneic.
 36. Amethod according to claim 1, wherein the T cell population is selectedor expanded from a mobilised apheresis sample.
 37. A method according toclaim 1, wherein the T cell population is selected usingFab-streptamers.
 38. A method according to claim 1, wherein the T cellpopulation is administered by infusion.